技术资料

Neutrophils are essential innate immune cells with unusual anti-microbial properties while dysfunctions of neutrophils lead to severe health problems such as lethal infections. Generation of neutrophils from human induced pluripotent stem cells (hiPSCs) is highly promising to produce off-the-shelf neutrophils for transfusion therapies. However,the anti-microbial potencies of hiPSCs derived neutrophils (iNEUs) remain less documented. Here,we develop a scalable approach to generate iNEUs in a chemical defined condition. iNEUs display typical neutrophil characters in terms of phagocytosis,migration,formation of neutrophil extracellular traps (NETs),etc. Importantly,iNEUs display a strong killing potency against various bacteria such as K.pneumoniae,P.aeruginosa,E.coli and S.aureus. Moreover,transfusions of iNEUs in mice with neutrophil dysfunction largely enhance their survival in lethal infection of different bacteria. Together,our data show that hiPSCs derived neutrophils hold strong anti-microbial potencies to protect severe infections under neutrophil dysfunction conditions.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13619-025-00227-z. View Publication

IntroductionAccumulating evidence indicates that the increased presence of astrocytes is fundamentally linked to the neurological dysfunctions observed in individuals with Down syndrome (DS). REST (RE1-silencing transcription factor),as a chromatin modifier,regulates 15,450 genes in humans. REST is a key regulatory element that governs astrocyte differentiation,development,and the maintenance of their physiological functions. The downregulation of REST may disrupt the homeostatic balance of astrocytes in DS.MethodsThis study aims to elucidate the role of REST in DS-astrocytes through comprehensive transcriptomic analysis and experimental validation.ResultsTranscriptomic analysis identified that REST-targeted differentially expressed genes (DEGs) in DS astrocytes are enriched in pathways associated with inflammatory response. Notably,our findings in astrocytes derived from DS human induced pluripotent stem cells (hiPSCs) show that the loss of nucleus REST leads to an upregulation of inflammatory mediators and markers indicative of the presence of reactive astrocytes. Lithium treatment,which restored nucleus REST in trisomic astrocytes,significantly suppressed the expression of these inflammatory mediators and reactive astrocyte markers.DiscussionThese findings suggest that REST is pivotal in modulating astrocyte functionality and reactivity in DS. The loss of REST in DS-astrocytes prompts the formation of reactive astrocytes,thereby compromising central nervous system homeostasis. Lithium treatment possesses the potential to rescue astrocyte reactivity in DS by restoring nucleus REST expression. View Publication

Background?-catenin,acting as the core effector of canonical Wnt signaling pathway,plays a pivotal role in controlling lineage commitment and the formation of definitive endoderm (DE) during early embryonic development. Despite extensive studies using various animal and cell models,the ?-catenin-centered regulatory mechanisms underlying DE formation remain incompletely understood,partly due to the rapid and complex cell fate transitions during early differentiation.ResultsIn this study,we generated new CTNNB1-/- human ES cells (hESCs) using CRISPR-based insertional gene disruption approach and systematically rescued the DE defect in these cells by introducing various truncated or mutant forms of ?-catenin. Our analysis showed that a truncated ?-catenin lacking both N- and C-terminal domains (?N148C) could robustly rescue the DE formation,whereas hyperactive ?-catenin mutants with S33Y mutation or N-terminal deletion (?N90) had limited ability to induce DE lineage. Notably,the ?N148C mutant exhibited significant nuclear translocation that was positively correlated with successful DE rescue. Transcriptomic analysis further uncovered that two weak ?-catenin mutants lacking the C-terminal transactivation domain (CTD) activated primitive streak (PS) genes,whereas the hyperactive ?-catenin mutants activated mesoderm genes.ConclusionOur study uncovered an unconventional regulatory function of ?-catenin through weak transactivation,indicating that the levels of ?-catenin activity determine the lineage bifurcation from mesendoderm into endoderm and mesoderm.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13578-024-01279-5. View Publication

BackgroundMale factor infertility (MFI) is responsible for 50% of infertility cases and in 15% of the cases sperm is absent due to germ cell aplasia. Human induced pluripotent stem cell (hiPSC)-derived spermatogonial stem cells (hSSCs) could serve as an autologous germ cell source for MFI in patients with an insufficient sperm yield for assisted reproductive technology (ART). The endocannabinoid system (ECS) has been implicated to play a role in mouse embryonic stem cells (mESCs) and the human testicular environment. However,the contribution of the ECS in hiPSCs and hiPSC-derived hSSCs is currently unknown. Here,we aimed to assess whether hiPSCs and hiPSC-derived hSSCs are regulated by components of the ECS and whether manipulation of the ECS could increase the yield of hiPSC-derived SSCs and serve as an autologous cell-based source for treatment of MFI.MethodsWe reprogrammed human dermal fibroblasts (hDFs) to hiPSCs,induced differentiation of hSSC from hiPSCs and evaluated the presence of ECS ligands (AEA,2-AG) by LC/MS,receptors (CB1R,CB2R,TRPV1,GPR55) by qPCR,flow cytometry and immunofluorescent labeling. We then examined the efficacy of endogenous and synthetic selective ligands (ACPA,CB65,CSP,ML184) on proliferation of hiPSCs using real-time cell analysis (RTCA) and assessed the effects of on CB2R agonism on hiPSC pluripotency and differentiation to hSSCs.ResultshiPSCs from hDFs expressed the pluripotency markers OCT4,SOX2,NANOG,SSEA4 and TRA-1-60; and could be differentiated into ID4+,PLZF?+?hSSCs. hiPSCs and hiPSC-derived hSSCs secreted AEA and 2-AG at 10??10 ??10??9 M levels. Broad expression of all ECS receptors was observed in both hiPSCs and hiPSC-derived hSSCs,with a higher CB2R expression in hSSCs in comparison to hiPSCs. CB2R agonist CB65 promoted proliferation and differentiation of hiPSCs to hiPSC-hSSCs in comparison to AEA,2-AG,ACPA,CSP and ML184. The EC50 of CB65 was determined to be 2.092?×?10??8 M for support of pluripotency and preservation of stemness on hiPSCs from 78 h. CB65 stimulation at EC50 also increased the yield of ID4?+?hSSCs,PLZF?+?SSPCs and SCP3?+?spermatocytes from day 10 to 12.ConclusionsWe demonstrated here for the first time that stimulation of CB2R results in an increased yield of hiPSCs and hiPSC-derived hSSCs. CB65 is a potent CB2R agonist that can be used to increase the yield of hiPSC-derived hSSCs offering an alternative source of autologous male germ cells for patients with MFI. Increasing the male germ/stem cell pool by CB65 supplementation could be part of the ART-associated protocols in MFI patients with complete germ cell aplasia.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40659-025-00596-4. View Publication

Rare coding mutations cause ~45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases,but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs),we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity,often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context,four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing,we used the MPRA data to develop a regression model,EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together,we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD. View Publication

Nuclear speckles are membraneless organelles that associate with active transcription sites and participate in post-transcriptional mRNA processing. During the cell cycle,nuclear speckles dissolve following phosphorylation of their protein components. Here,we identify the PP1 family as the phosphatases that counteract kinase-mediated dissolution. PP1 overexpression increases speckle cohesion and leads to retention of mRNA within speckles and the nucleus. Using APEX2 proximity labeling combined with RNA-sequencing,we characterize the recruitment of specific RNAs. We find that many transcripts are preferentially enriched within nuclear speckles compared to the nucleoplasm,particularly chromatin- and nucleus-associated transcripts. While total polyadenylated RNA retention increases with nuclear speckle cohesion,the ratios of most mRNA species to each other are constant,indicating non-selective retention. We further find that cellular responses to heat shock,oxidative stress,and hypoxia include changes to the phosphorylation and cohesion of nuclear speckles and to mRNA retention. Our results demonstrate that tuning the material properties of nuclear speckles provides a mechanism for the acute control of mRNA localization. Here the authors study how interactions with nuclear speckles help localize mRNA in cells. They find that modifications of the proteins in these structures affects their cohesion and can modulate mRNA retention under stress. View Publication

PurposeLittle is known about the development of Bruch's membrane (BrM),the structure separating and supporting the retina and choroid,nor whether differentiation of human pluripotent stem cell (hPSC)-derived retinal pigment epithelium (RPE) accurately replicates BrM. This has relevance for tissue engineering strategies,both in the development of accurate in vitro models,and effective RPE transplant strategies. Here,we investigated BrM-associated protein production in human fetal tissue and hPSC-derived RPE.MethodsThe presence of laminin,elastin,fibronectin,and types I/III/IV collagen was examined in human fetal eyes at 6 to 21 post-conception weeks (PCWs) and hPSC-derived RPE cultures at 1 to 6 weeks in culture using immunohistochemistry/immunocytochemistry and quantitative PCR (qPCR).ResultsIn human fetal retina,laminin and fibronectin were present from 6 PCW,type IV collagen from 8 PCW,elastin from 12 PCW,type I collagen by 17 PCW,and type III collagen from 21 PCW. BrM layering was discernible from 12 PCW,becoming distinct by 17 PCW. In hPSC-derived RPE cultures,basement membranes containing laminin and fibronectin were present from week 1,type IV collagen from week 2,and type I collagen from week 4. Type III collagen was present at all timepoints,although not localized as a basement membrane. Elastin was absent at all timepoints.ConclusionsBrM-like membrane synthesis in hPSC-derived RPE largely recapitulates the temporal sequence seen in human development,excluding elastin. These support the utility of hPSC-derived RPE in in vitro systems to model RPE/retina interactions in health and disease,and inform cell therapy approaches,as de novo BrM-like membrane has the potential to support transplanted donor RPE. View Publication

Human pluripotent stem cells (hPSCs) and their differentiated derivatives represent valuable tools for studying development,modeling diseases,and advancing cell therapy. Recent improvements in genome engineering allow for precise modifications of hPSCs,further enhancing their utility in basic and translational research. Here we describe a Recombinase-Mediated Cassette Exchange (RMCE) platform in hPSCs that allows for the highly efficient,rapid,and specific integration of transgenes. The RCME-mediated DNA integration process is nearly 100% efficient,without negatively affecting the pluripotency or karyotypic stability of hPSCs. Taking advantage of this convenient system,we first established a dual inducible expression system based on the Tet-On and Cumate-On systems,allowing for the inducible expression of two transgenes independently. Secondly,we incorporated a Tet-on inducible system,driving the expression of three genes simultaneously. However,two genes also contain independent degron sequences,allowing for precise control over the expression of each gene individually. We demonstrated the utility of these systems in hPSCs,as well as their functionality after differentiation into cells that were representative of the three germ layers. Lastly,we used the triple inducible system to investigate the lineage commitment induced by the pancreatic transcription factors NKX6.1 and PDX1. We found that controlled dual expression,but not individual expression,biases hPSC embryoid body differentiation towards the pancreatic lineage by inducing the expression of the NeuroD program. In sum,we describe a novel genetic engineering platform that allows for the efficient and fast integration of any desired transgene(s) in hPSCs using RMCE. We anticipate that the ability to modulate the expression of three transgenes simultaneously will further accelerate discoveries using stem cell technology. View Publication

Dysregulation of mitochondrial function has been implicated in Parkinson’s disease (PD),but the role of mitochondrial metabolism in disease pathogenesis remains to be elucidated. Using an unbiased metabolomic analysis of purified mitochondria,we identified alterations in ?-ketoglutarate dehydrogenase (KGDH) pathway upon loss of PD-linked CHCHD2 protein. KGDH,a rate-limiting enzyme complex in the tricarboxylic acid cycle,was decreased in CHCHD2-deficient male mouse brains and human dopaminergic neurons. This deficiency of KGDH led to elevated ?-ketoglutarate and increased lipid peroxidation. Treatment of CHCHD2-deficient dopaminergic neurons with lipoic acid,a KGDH cofactor and antioxidant agent,resulted in decreased levels of lipid peroxidation and phosphorylated ?-synuclein. CHCHD10,a close homolog of CHCHD2 that is primarily linked to amyotrophic lateral sclerosis/frontotemporal dementia,did not affect the KGDH pathway or lipid peroxidation. Together,these results identify KGDH metabolic pathway as a targetable mitochondrial mechanism for correction of increased lipid peroxidation and ?-synuclein in Parkinson’s disease. An unbiased metabolomic analysis identifies ?-ketoglutarate dehydrogenase metabolic pathway as a targetable mitochondrial mechanism for correction of increased lipid peroxidation in CHCHD2-linked Parkinson’s disease models. View Publication

Viral vector delivery of gene therapy represents a promising approach for the treatment of numerous retinal diseases. Adeno-associated viral vectors (AAV) constitute the primary gene delivery platform; however,their limited cargo capacity restricts the delivery of several clinically relevant retinal genes. In this study,we explore the feasibility of employing high-capacity adenoviral vectors (HC-AdVs) as alternative delivery vehicles,which,with a capacity of up to 36 kb,can potentially accommodate all known retinal gene coding sequences. We utilized HC-AdVs based on the classical adenoviral type 5 (AdV5) and on a fiber-modified AdV5.F50 version,both engineered to deliver a 29.6 kb vector genome encoding a fluorescent reporter construct. The tropism of these HC-AdVs was evaluated in an induced pluripotent stem cell (iPSC)-derived human retinal organoid model. Both vector types demonstrated robust transduction efficiency,with sustained transgene expression observed for up to 110 days post-transduction. Moreover,we found efficient transduction of photoreceptors and Müller glial cells,without evidence of reactive gliosis or loss of photoreceptor cell nuclei. However,an increase in the thickness of the photoreceptor outer nuclear layer was observed at 110 days post-transduction,suggesting potential unfavorable effects on Müller glial or photoreceptor cells associated with HC-AdV transduction and/or long-term reporter overexpression. These findings suggest that while HC-AdVs show promise for large retinal gene delivery,further investigations are required to assess their long-term safety and efficacy. View Publication

A distinctive feature of both type 1 and type 2 diabetes is the waning of insulin-secreting beta cells in the pancreas. New methods for direct and specific targeting of the beta cells could provide platforms for delivery of pharmaceutical reagents. Imaging techniques such as Positron Emission Tomography (PET) rely on the efficient and specific delivery of imaging reagents,and could greatly improve our understanding of diabetes etiology as well as providing biomarkers for viable beta-cell mass in tissue,in both pancreas and in islet grafts.The DiGeorge Syndrome Critical Region Gene 2 (DGCR2) protein has been suggested as a beta-cell specific protein in the pancreas,but so far there has been a lack of available high-affinity binders suitable for targeted drug delivery or molecular imaging. Affibody molecules belong to a class of small affinity proteins with excellent properties for molecular imaging. Here,we further validate the presence of DGCR2 in pancreatic and stem cell (SC)-derived beta cells,and then describe the generation and selection of several Affibody molecules candidates that target human DGCR2. Using an in-house developed directed evolution method,new DGCR2-binding Affibody molecules were generated and evaluated for thermal stability and affinity. The Affibody molecules variants were further developed as targeting agents for delivering imaging reagents to beta cell. The Affibody molecule ZDGCR2:AM106 displayed nanomolar affinity,suitable stability and biodistribution,with negligible toxicity to islets,qualifying it as a suitable lead candidate for further development as a tool for specific delivery of drugs and imaging reagents to beta cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-84574-y. View Publication

AbstractThe generation of chimeric antigen receptor?modified natural killer (CAR?NK) cells using induced pluripotent stem cells (iPSCs) has emerged as one of the paradigms for manufacturing off?the?shelf universal immunotherapy. However,there are still some challenges in enhancing the potency,safety,and multiple actions of CAR?NK cells. Here,iPSCs were site?specifically integrated at the ribosomal DNA (rDNA) locus with interleukin 24 (IL24) and CD19?specific chimeric antigen receptor (CAR19),and successfully differentiated into iPSC?derived NK (iNK) cells,followed by expansion using magnetic beads in vitro. Compared with the CAR19?iNK cells,IL24 armored CAR19?iNK (CAR19?IL24?iNK) cells showed higher cytotoxic capacity and amplification ability in vitro and inhibited tumor progression more effectively with better survival in a B?cell acute lymphoblastic leukaemia (B?ALL) (Nalm?6 (Luc1))?bearing mouse model. Interestingly,RNA?sequencing analysis showed that IL24 may enhance iNK cell function through nuclear factor kappa B (NF?B) pathway?related genes while exerting a direct effect on tumor cells. This study proved the feasibility and potential of combining IL24 with CAR?iNK cell therapy,suggesting a novel and promising off?the?shelf immunotherapy strategy. Zhang et al. successfully regenerated iNK cells from human iPSCs with rDNA locus gene editing. IL24 enhances the antitumor activity and proliferation of armored CAR?iNK cells,which may be involved in cellular?positive upregulation and adhesion pathways. View Publication